Limitations Pulse-Modulated Technique Measuring ... · depends on the history of the sample and the conditions underwhichthe measurementsare conducted(10, 13). Utilization of the

Plant Physiol. (1992) 100, 367-3730032-0889/92/100/0367/07/$01 .00/0

Received for publication March 5, 1992Accepted May 4, 1992

Limitations of the Pulse-Modulated Technique for Measuringthe Fluorescence Characteristics of Algae1

Claire S. Ting* and Thomas G. Owens

Section of Plant Biology, Cornell University, Ithaca, New York 14853-5908

ABSTRACT

Precise measurements of the minimal fluorescence yield (F.) andmaximal fluorescence yield (Fm) of a dark-adapted sample areprerequisites for the quantification of other fluorescence parame-ters. The pulse amplitude-modulated chlorophyll fluorometer (PAM101 Chlorophyll Fluorometer, Heinz Walz, Effeltrich, Germany)and saturating pulse technique have frequently been used in meas-uring F. and Fm and in resolving the contributions of photochemicaland nonphotochemical quenching to the total fluorescence yield.The extent to which instrument-dependent factors may affect theaccurate measurement of F. and Fm is addressed. It is shown thatthe increase in pulse amplitude-modulated measuring beam inten-sity at 1.6 and 100 kHz was nonlinear at higher light intensitysettings. The implications of this for measurements of F. (1.6 kHz)and Fm (100 kHz) are discussed. It is also demonstrated thatunderestimation of Fm may result due to saturation of the PAM 101photodiode by scattered infrared light associated with intense lightpulses. In addition, it is shown how sample-dependent factors mayaffect measurements of F. and Fm in samples with low chlorophyllconcentrations, in particular, dilute algal suspensions of Phaeodac-tylum tricornutum and Chiamydomonas reinhardtii. A technique ispresented for the accurate measurement of F. in algal suspensions(<8 gg chlorophyll a mL-1). The importance of examining thesaturating pulse transient and Fm level as a function of the dampingsetting, pulse width, and pulse intensity, and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea is discussed.

The quantitative analysis of Chl a fluorescence quenchinghas provided information on processes that may be regulatingPSII photochemistry during steady-state photosynthesis (7,13, 16, 19). Quenching of Chl fluorescence has been attrib-uted to both photochemical (qp2) and nonphotochemical (qN)processes. qp reflects the competition between photochemis-try and fluorescence for deactivation of Chl-excited states inthe antenna. When photochemistry is blocked due to reduc-

'This work was supported by a grant to T.G.O. from the NationalScience Foundation (No. DMB-8803626) and a Hatch Grant (No.185405). C.T. was supported in part by a Lewis Knudson Fellowship(Cornell University).

2 Abbreviations: qp, photochemical fluorescence quenching; qN,nonphotochemical fluorescence quenching; F0, minimal fluorescenceyield of a fully dark-adapted sample; Fo', minimal fluorescence yieldin any light-adapted state; Fm, maximal fluorescence yield of a fullydark-adapted sample; Fm', maximal fluorescence yield in any light-adapted state; LED, light-emitting diode; PAM, pulse amplitudemodulated; QA, primary quinone acceptor of PSII; sp, saturatingpulse.

tion of the primary quinone acceptor (closed trap), photo-chemical quenching of excited states is eliminated (qp = 0)and the fluorescence yield is maximal. When all reactioncenters are capable of photochemistry, qp is maximal (=1)and the fluorescence yield is low. In contrast, a diverse groupof components contributes to qN; these essentially reflect thenonphotochemical pathways by which excited states in theantenna can be deactivated. The primary component involvesthermal deactivation of excited states and is associated mainlywith the establishment of the transthylakoid pH gradientduring electron transport and possibly with the xanthophyllcycle (2, 12). Other qN components include state transitionsand photoinhibition (4, 6, 9, 15). The extent to which eachcomponent contributes to the total amount of qN measureddepends on the history of the sample and the conditionsunder which the measurements are conducted (10, 13).

Utilization of the PAM Chl fluorometer (PAM 101 Chlo-rophyll Fluorometer, Heinz Walz, Effeltrich, Germany) (16)and the saturating light pulse (light-doubling) technique (1)have proven useful in resolving the contributions of both qPand qN to the fluorescence yield observed under differentphysiological conditions. The rationale behind this techniqueis that application of a brief (0.02-2.0 s) high-intensity pulseof light to a sample will result in the transient reduction ofthe primary electron acceptor QA without affecting the proc-esses determining qN. The corresponding rise in fluorescenceyield reflects the temporary removal of all qP. Any remainingquenching below Fm is assigned to qN, but does not specifythe mechanism(s) causing this quenching. Although this tech-nique can yield valuable information about the photosyn-thetic physiology of the sample, instrument complicationsand incorrect assumptions about the physiological quenchingstate of the sample may affect the accuracy of fluorescencemeasurements and their interpretation. In this article, wedemonstrate the extent to which instrument-dependent fac-tors may affect the accurate measurement of F. (qp = 1, qN =0) and Fm (qp = 0, qN = 0). The precise measurement of thesetwo parameters is particularly important because all otherfluorescence parameters are normalized between these twovalues. In addition, we will show how sample-dependentfactors may affect measurements of fluorescence parametersin samples with low (<8 ,M) Chl concentration, in particular,dilute algal suspensions.

MATERIALS AND METHODS

Chlamydomonas reinhardtii was grown at 200C and 25 ,Em-2 s-1 in Tris acetate phosphate medium (17), whereas

367

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Plant Physiol. Vol. 100, 1992

Phaeodactylum tricornutum was grown at 150C and 100 gEm-2 s-1 in natural seawater containing "f/2" nutrients (3).Unialgal axenic batch cultures (2 L) of both species were

maintained in the early log phase of growth in order tominimize self-shading. Chl a was determined in 90% (v/v)acetone spectrophotometrically (8).

Fluorescence measurements were conducted at 20 and150C for Chlamydomonas and Phaeodactylum, respectively,using a PAM Chl fluorometer. Fluorescence data collectionwas achieved by interfacing the PAM fluorometer to a Hew-lett Packard series 200 computer (200-kHz analog/digitalconverter, Infotek, Anaheim, CA). Samples were enclosed ina Hansatech DW2 oxygen electrode chamber (1.5 mL)equipped with an adapter for the PAM fluorometer. Withwhole cell samples, cells were dark-adapted for 5 min priorto initial (F.) and maximum (Fm) fluorescence level measure-

ments. Unless otherwise noted, nomenclature for fluores-cence parameters is as defined in reference 18. F. levels weresampled at 20-ms intervals for a total of 20 s and thenaveraged. F. and Fm measurements were conducted at instru-ment frequency settings of 1.6 and 100 kHz, respectively. Fmvalues represent the maximum fluorescence level measuredduring the last 1.0 s (1000 data points collected at 1-msintervals and averaged) of a 1.5-s high-intensity light pulse(6800 ,uE m-2 s-1, blue light excitation) delivered to thesample. The saturating light pulse source was a 1000-Wtungsten-halogen lamp blocked either by a Corning 4-94filter for blue light excitation, a Kodak 88A long-pass filter(cutoff wavelength at 720 nm) for IR excitation, or a 3-cmwater bath and two heat-absorbing filters for white lightexcitation. Duration of illumination was controlled by an

electronic shutter (Uniblitz, Rochester, NY) with an effectiveopening time of 5 ms. For comparison, Fm was also deter-mined in the presence of 50 ,lM DCMU using either a

saturating light pulse (6800 ,uE m-2 s-1 blue light, Fm [sp,DCMU]) or background illumination (130 ,uE m-2 s-1 whitelight, Fm [actinic, DCMU]) to close the traps. DCMU was

added in the dark after Fo determination and 5 min prior tothe measurement of Fm. Numerical values reported here are

in volts.Chl was extracted from Chlamydomonas in 90% (v/v) ace-

tone by using a tissue homogenizer to rupture cells concen-

trated on glass fiber filters. These extracts were utilized inexamining the signal output of the PAM Chl fluorometer atinstrument frequency settings of both 1.6 and 100 kHz, andat different measuring beam intensities. To characterize theinstrument response to high-intensity light pulses, fluores-cence levels of Chl extracts were monitored in the absenceand presence of high-intensity (7200 uE m-2 s-1, white light)pulses. Intensities were attenuated by adding neutral densityfilters. In experiments where the effect of the IR componentof the saturating light pulse source was examined, IR excita-tion was used instead of white light. Light intensity was

measured at a distance of about 1 mm from the end of thePAM fiber optics cable using a calibrated IL1700 ResearchRadiometer/Photometer equipped with a PAR No. 10016filter or a F No. 9988 filter for PAR (,uE m-2 s-1) and IR (Wm-2) measurements, respectively.

RESULTS AND DISCUSSION

Pulsed Measuring Light Intensity and Frequency Settings

Knowledge of the intensity of the pulsed LED measuringbeam of the PAM fluorometer is useful for several reasons.

First, in the absence of any actinic effects, the amplitude ofthe fluorescence signal is directly proportional to the meas-

uring beam intensity. Thus, the PAM measuring beam inten-sity is the primary factor in determining the signal to noiseratio of a measurement. Second, proper measurements of Foand F.' require that the measuring beam have a minimumactinic effect, again a function of measuring intensity. Finally,accurate determination of F. (1.6 kHz) and Fm (100 kHz)requires that the measuring intensities scale properly betweenthe two modulation frequencies. These factors were examinedbelow.The 12 different intensities of the pulsed measuring beam

at 1.6 and 100 kHz are presented in Table I. At each instru-ment light intensity setting (1-12), the intensity of the meas-

uring beam is increased by an average factor of 60.11 between1.6 and 100 kHz; this is close to the expected increase of62.50 (100/1.6 kHz). However, Table I also indicates that as

one progresses to higher instrument light intensity settings,the increase in intensity at 1.6 kHz is slightly larger than theincrease at 100 kHz; this results in the decrease in the ratioof light intensity at 100 to 1.6 kHz at higher intensity settings.Factors which may account for this include LED outputlimitations such as decreases in emission yields at higherpulse currents and frequencies due to localized heating withinthe LED (PAM Chlorophyll Fluorometer System Description,Operation Manual and Examples of Application, Heinz Walz,Germany).As a result of variations in excitation intensity between 1.6

and 100 kHz, one would expect that the ratio of fluorescencesignals at 1.6 and 100 kHz would also vary with PAMintensity setting. This was demonstrated experimentally inFigure 1 for fluorescence from Chl extracts. The ratio offluorescence at 100 to 1.6 kHz ranged from 1.4 (setting = 1)

Table I. Intensities of the PAM Measuring Beam at 1.6 and 100 kHzMeasuring beam intensities were measured at each instrument

light intensity setting at a distance of about 1 mm from the end ofthe PAM fiber optics cable. Ratios of the intensities at 100 to 1.6kHz at the same instrument setting are also presented.

PAM Light Light Intensity Light Intensity Ratio ofIntensity Setting at 1.6 kHz at 100 kHz 100/1.6 kHz

ME m2 s-' AE m2 s-l1 0.0006 0.0358 61.72 0.0012 0.0757 61.53 0.0026 0.1581 61.04 0.0051 0.309 60.75 0.0092 0.558 60.86 0.015 0.926 60.87 0.026 1.60 60.68 0.042 2.51 60.29 0.061 3.62 59.6

10 0.080 4.72 58.911 0.101 5.86 58.112 0.125 7.16 57.2

368 TING AND OWENS



CHL FLUORESCENCE MEASUREMENTS IN ALGAE

1.4 * Fluorescence ratio (-) scatterer

0 Fluorescence ratio(.) scatterer

0 o 1.2

0 0

:J00

0.91 2 3 4 5 6 7 8 9 10 11 12

PAM 101 Light Intensity Setting

Figure 1. The ratio of fluorescence measured at 100 kHz to fluo-rescence measured at 1.6 kHz as a function of PAM 101 lightintensity setting for pure Chl (0) (14 Mg Chl a mLV1). 0, In thepresence of scatterer (magnesium carbonate). A, The ratio of theintensity of the PAM measuring beam at 100 to 1.6 kHz for eachPAM light intensity setting (1-12) normalized to the value of thisratio at a light intensity setting of 6 (60.8).

to 0.93 (setting = 12) and was independent of the presence

of scatterer and Chl concentration. This decrease can beaccounted for almost entirely by the decrease in the ratio ofthe intensity of the pulsed measuring beam at 100 to 1.6 kHzat higher light intensity settings (Table I). A plot of this ratioat each light intensity setting normalized to the ratio at a

setting of 6 (60.8, Table I) parallels the decline in the ratio offluorescence yields, especially at higher intensity settings (Fig.1). For the yields of fluorescence at F. (1.6 kHz) and Fm (100kHz) to be comparable, instrument light intensity settingswhere this fluorescence ratio is 1 should be used or valuesmust be scaled accordingly. Accurate measurements of bothF. and Fm are particularly important because other Chl fluo-rescence parameters are normalized between these twovalues.

Time Constants of Instrument Damping Settings

The electrical time constant (defined as the time to complete63% of the maximum signal change) of each damping settingof the PAM fluorometer is presented in Tables II and III.Differences that exist between these values and those pub-lished in the PAM Instruction Manual cannot be accounted

Table II. Functional Time Constants Associated with PAM DampingSettings of 0 to 4

Fast time constants were resolved by delivering a single turnoverflash (<1 Ms rise time, approximately 1 Mis pulse width) to the sampleand monitoring the rate of the fluorescence rise from Fo to Fm on a

storage oscilloscope.Damping Setting Time Constant

0 2 As1 3 As2 4 s3 7Ms4 54,us

Table HIL. Functional Time Constants Associated with PAMDamping Setting of 5 to 11Time constants were determined by delivering a saturating light

pulse (0.02-1.5 s pulse width, blue light excitation) to the sampleand measuring the rate of fluorescence decay from Fm to F,The effective opening time (<5 ms) of the electronic shutterused in controlling the pulse width was accounted for in thesemeasurements.

Damping Setting Time Constant

5 7 ms6 11 ms7 20 ms8 41 ms9 122 ms

10 255 ms1 1 965 ms

for because details are not provided about how the dampingtime constants in the Instruction Manual were measured.However, we determined the time constants under experi-mental conditions in which each setting would normally beused. A functional time constant for each damping setting(0-11) on the PAM fluorometer was determined by measur-

ing the rate of fluorescence decay from Fm to F. followingeither single tumover flashes (EG&G Xenon Flash LampModel FX-201; <1 ,us rise time, 1 Ms pulse width, settings 0-4) or saturated light pulses (0.02-1.5 s, blue light, settings 5-11). The effects of the 5-ms shutter opening/closing timewere taken into account in these latter measurements. TablesII and III indicate that the time constants of the dampingsettings range from 2 ,s to 965 ms. Each time constantessentially establishes the maximum rate of change in thesignal output from the instrument. This allows for the elimi-nation of any background noise that varies at a faster ratethan the time constant of the damping setting being used.However, to avoid the incorrect damping of fluorescencesignals, settings should be used which have time constantsthat are faster than the fluorescence changes of interest.Utilization of settings with time constants that are larger than

Table IV. PAM Gain SettingsThe relative gain and incremental gain in the amplitude of the

fluorescence signal as a function of PAM gain setting.PAM Setting Relative Gain Incremental Gain

1 1 12 3 33 5 1.674 7 1.405 9 1.296 1 1 1.227 13 1.188 15 1.159 17 1.1310 19 1.1211 21 1.1112 13 1.10

369




1000 2000 3000 4000 5000 6000 7000

Saturating Pulse Intensity (PAR uE mn )

90

80

70 P

50°

40-i

+

30B

20'-

10O)0 rA4~~

00a.

CS

4.O

ad

C)

te

So

0-

Si

Figure 2. Dependence of the ratio of fluorescence in the presenceof a saturating light pulse (F,p) to fluorescence in the absence of a

saturating light pulse (Fsp) on pulse intensity for pure Chl (8 lsg chla ml-') in the absence (A) and presence (0) of a scatterer (magne-sium carbonate). Saturating pulse beams were blocked by a waterbath and two heat absorbing filters. Unfilled circles represent theamount of energy (W m-2) due to wavelengths greater than 720 nm(saturating light pulse beam blocked by a Kodak 88A long-passfilter) present at each saturating light pulse intensity.

the rate of the expected fluorescence changes will result indata loss.

PAM Gain Settings

Increases in the amplitude of the fluorescence signal are

often required when working with samples with low fluores-cence yields. The signal can in part be amplified by increasingthe instrument gain setting (positions 1-12). Values of boththe relative gain and incremental gain in the amplitude ofthe fluorescence signal at each instrument setting are pre-sented in Table IV.

Instrument Response to High-intensity Light Pulses

Utilization of the "light-doubling" technique (1) to separateqp and qN requires the use of short (0.02-2 s) high-intensitylight pulses for the transient reduction of QA. However,increases in the fluorescence signal or scattered IR lightassociated with the use of intense light pulses may lead tosaturation of the PAM 101 photodiode. To ensure that arti-facts caused by saturation of the photodiode are absent, weexamined instrument responses to these pulses. Chl extractswere first used to examine the instrument signal output inresponse to short, high-intensity (7600 MuE m-2 s-', whitelight) pulses. With these extracts, one would expect thefluorescence yield measured by the modulated measuringbeam to be the same in the presence (F+sp) or absence (F-sp)of the saturating light pulse. However, we observed a con-

sistent decline in fluorescence levels with increasing pulseintensity (Fig. 2). This phenomenon was independent of thepresence of scatterer, Chl concentration, and cuvette geom-

etry. These data suggested that the use of intense light pulsesmay cause saturation of the amplifier system of the PAM 101detector unit, resulting in underestimation of Fm and Fm'.To examine the potential contributions of scattered IR light

Figure 3. Dependence of the ratio of fluorescence in the presence

of a saturating light pulse (F+sp) to fluorescence in the absence of asaturating light pulse (F5p) on pulse intensity for pure Chi (8 ,tg Chla mL-) in the absence (-) and presence (A) of a scatterer (magne-sium carbonate). A Kodak 88A long-pass filter was used to blockthe saturating light source, and all high light intensity pulses deliv-ered to the sample were of wavelengths greater than 720 nm.

to saturation of the photodiode, a Kodak 88A long-pass filter(cutoff wavelength at 720 nm) was used in the path of thehigh-intensity light source, and IR light pulses of differentintensities were delivered to the Chl sample. We observedthat as the amount of IR light in the saturating light pulseincreased, there was a continuous decline in the fluorescencelevel. At approximately 350W m-2 IR, the fluorescence signalvanished due to saturation of the photodiode (Fig. 3). Al-though the PAM 101 photodiode is blocked by a RG-9 long-pass filter and is thus protected against reflected and scatteredlight of wavelengths less than 700 nm, silicon photodiodedetectors have their peak sensitivity at 800 nm and theirdetection capacity extends beyond 1 ,um. In Figure 2, the 5%decrease in F+sp/F0,p at 7600 ,E m-2 s-1 (PAR), which contains70 W m-2 of IR light (wavelengths > 720 nm), corresponds

4.0 FA

-

arZ.

"I,

3.5 [

3.0

2.5 F

2.0

0.30

0.25 ot

0.20 0

0.15 pi

'ti

0.10 _-

0

0.05 %.

0 10 20 30 40 50 60 70 80 90 100-I

Chlorophyll a Concentration (ug Chl a ml )

Figure 4. Changes in the ratio of Fm/F. (A), F. (0), and Fm (0) as a

function of Chl concentration in Chlamydomonas. Cells were dark-adapted for 5 min prior to measurements. A Corning 4-94 blue-green filter was used to block the saturating light pulse beam (6800,uE m-2 s-1) during measurements of Fm.

0

d

CO

+-

dCa)

C)asa

a.0

0

aIL

S

19nO

S

0

S1

100 200 300 400 500 600 700 800 900IR Saturating Pulse Intensity (> 720 am, W m )

I I

;> *-00

370 TING AND OWENS




Table V. Comparison of Fluorescence Parameters Measured inChlamydomonas at 10 Mg Chl a/mL and 50 Mg Chl a/mL

Instrument settings and conditions were identical between meas-urements. All samples were dark-adapted for 5 min prior to meas-urements. Fm levels were measured using a 6800 ME m-2 S-1 (bluelight) saturating light pulse.

[Chl a] F. Fm Fm/Fo Fo.o,g/mtl Fm.o,g/mL

Mg ml/l10 0.0441 0.1796 4.0750 0.1141 0.3031 2.66 2.59 1.69

to the 5% decrease observed in this same ratio at 70 W m-2(wavelengths > 720 nm) in Figure 3. Therefore, the decreaseobserved in this ratio in Figure 2 is attributable to IR satura-tion of the photodiode. From these results, we conclude thatutilization of typical heat-absorbing filters and water bathswill not prevent IR saturation of the PAM 101 photodiodewhen high-intensity light pulses are used.

Measurements of F. and Fm in Algal Suspensions

At low Chl concentrations, noise limitations of the PAMfluorometer complicate fluorescence measurements. In orderto maximize signal to noise ratios when working with algalsuspensions, one approach has been to increase the overallfluorescence signal by using high concentrations (>10 ,ug Chla mL-1) (5). However, our results suggest that this approachshould be avoided. Working with whole cell Chlamydomonassuspensions, we observed that the value of Fm/Fo was stronglydependent on Chl concentration (Fig. 4; Table V). Decreases

0.35 I

.30 Phaeodactylum>N05 ; (7 ugml)-0.25-

0.20-

0.15-

o 0.10

0 0.05

0.000.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

PAM Measuring Beam Intensity (uE m s )

Figure 5. Changes in the value of F. as a function of instrumentlight intensity setting (1-12) for whole cells of P. tricornutum (7 AgChi a mL-1). Instrument light intensity settings 4, 8, and 12 are

labeled on the plot. Samples were dark-adapted for 5 min prior tothe start of measurements. All measurements were conducted at1.6 kHz. A regression analysis was conducted on the first six datapoints (representing the first six light intensity settings of the PAM101 fluorometer), and the regression line is as shown on the graph.

.64..-0oqt-

U)a)

a)a0

I

r=4r-4

2000 4000 6000 8000 10000 12000

Saturating Pulse Intensity (PAR uE m s )

Figure 6. Fm measured as a function of saturating light pulse inten-sity for Chlamydomonas (d) and Phaeodactylum (0). Cell concentra-tions were 6 MAg Chi a mL?. Fm levels were measured during thedelivery of a 6800 ME m-2 s-' (blue light excitation, 1.5 s) light pulse,and each point represents the average of 1000 data points collectedat 1-ms intervals.

in this ratio were apparent at Chl concentrations greater than10 sg mL-1. These decreases were attributable to the fact thatincreases in Fo with Chl concentration were larger than theincrease in Fm. Disproportional changes may be due to theoptical properties of the stirred algal suspension. AlthoughF. and Fm are measured in the presence of a modulatedmeasuring beam, Fm measurements are made with the addi-tion of a high-intensity light source. Light attenuation by thestirred sample at high Chl concentrations may lead to a

nonuniform illumination of the algal suspension and inac-curate measurements of Fm levels. Therefore, raising the Chlconcentration to increase the signal to noise ratio will lead toinaccurate measurements of Fm/Fo.The correct measurement of Fm/Fo ratios in algal suspen-

sions has been problematic due to the low signal to noiseratio present during the F. measurement. This is primarilybecause Fo is measured at 1.6 kHz and at instrument light

Table VI. Saturating light pulse Intensities and Pulse Widths Usedto Completely Reduce QA

Saturating light pulse intensities and pulse widths used in deter-mining Fm by the light-doubling technique in different higher plantand green algal systems.

sp Intensity Used(and Pulse Width) System Reference

ME m-2 s-'500 Wheat, maize (7)750 (800 ms) Spinach (4)840 (1.2 s) Spinach, pea (12)1000 Green alga (Selenastrum) (11)1800 (1 s) Barley (6)1895 Spinach (10)3600 (700 ms) Barley (15)7500 (0.7 s) Tobacco (14)

371




Table VIl. Fluorescence Parameters Measured at 10 ,ug Chl a/mL inChlamydomonas (137c-) and Phaeodactylum (PT646)

All samples were dark-adapted for 5 min prior to measurements.Fm levels were measured as described in "Materials and Methods."Samples were mixed in the dark for 5 min following DCMU (50,lMfinal concentration) addition, and Fm (sp, DCMU) levels were deter-mined by the application of a saturating light pulse (6800 AE m-2s51, 1.5 s, blue light) to the sample.

Species Fo Fm Fm/Fo Fm (sp, DCMU)

137c- 0.0488 0.1929 3.95 0.1904PT646 0.0639 0.1630 2.55 0.1813

intensity settings of 7 or lower to avoid actinic effects of themeasuring beam. At measuring beam intensity settings of 7(0.026 ,uE m-2 s-', 1.6 kHz) or higher, the changes in the F.as a function of measuring beam intensity become nonlinear(Fig. 5). We examined the frequency of noise associated withour actual F. signal. For both Chlamydomonas and Phaeodac-tylum, the noise contributions were on the order of ±10 and±2 mV at sampling frequencies of 1.6 and 100 kHz, respec-

tively (light intensity = 7, gain = 8, damping = 8). At the1.6-kHz sampling frequency, we observed two time scales(milliseconds and seconds) of F. variability. When F, was

measured at 5-ms intervals (1000 data points collected andaveraged to obtain the final Fo level), the SD of 10 F. levelswas ±0.00113. However, when Fo was sampled at 20-msintervals (1000 data points collected and averaged to obtainthe final Fo level), the SD decreased to ±0.00052. The amountof variability associated with the F. measurement also in-creased to ±0.00117 when the total number of data pointscollected and averaged to obtain the final Fo level was reducedfrom 1000 (20 s total sampling time) to 100 (2 s total samplingtime). To improve the signal to noise ratio at 1.6 kHz, we

therefore decided to sample F. at 20-ms intervals and average

the result of 1000 data points to obtain the final F. level. Thisapproach results in reproducible Fo levels in algal suspensionsat Chl concentrations less than 8 ,ug Chl a mL-1.To measure Fm levels accurately using the light-doubling

technique, the shape of the saturating light pulse transientmust be examined as a function of the intensity of thesaturating light pulse, the pulse width, and the dampingsetting. These three parameters should be adjusted to givethe highest signal to noise ratio as well as saturation of thefluorescence yield rise. The shortest possible pulse lengthshould also be chosen to avoid secondary effects. In addition,Fm levels can be checked by measuring changes in Fm over a

range of pulse intensities. Plots of Fm versus saturating lightpulse intensity for Chlamydomonas and Phaeodactylum indi-cate that although light pulses of about 2000 /E m-2 s-' willcompletely reduce QA in Chlamydomonas, they will not inPhaeodactylum (Fig. 6). It clearly should not be assumed thata certain light intensity is necessarily saturating for all algalspecies because factors that affect the value of Fm, such as

the ratio of PSII to Cyt b6f to PSI and the size of the lightharvesting antenna complex and plastoquinone pool, vary

between species.Fm levels can also be checked by the addition of DCMU to

the sample. In the presence of either DCMU and backgroundactinic light or a high-intensity saturating light pulse, the Chla fluorescence level of a dark-adapted sample (qN = 0) isexpected to reach the same maximum level (Fm = Fm [sp,DCMU]). This Fm level reflects a state where QA is fullyreduced, and the amount of qp and qN fluorescence quenchingand the potential for photochemistry are at a minimum. At asaturating light pulse intensity of 6800 jE m-2 s-' (blue light),which is similar to or greater than that typically used tomeasure Fm in higher plants and green algae (Table VI), weobserved that Fm levels determined by both of these tech-niques were approximately the same for whole cells of Chlam-ydomonas (Fm = Fm [sp, DCMU]) (Table VII). However, inPhaeodactylum, the Fm (sp, DCMU) level was greater thanthat determined with the saturating light pulse alone (TableVII). Although Figure 6 suggests that a 6800 ,uE m-2 s-1 lightpulse should completely reduce QA in Phaeodactylum, if anynonphotochemical quenching were present in the dark-adapted state, fluorescence levels may not reach their truemaximum level until this quenching is relaxed. This possibil-ity will be addressed in a future paper. It is clear from theseresults that to measure Fm accurately in different algal species,measurement conditions must be carefully optimized for eachspecies.

LITERATURE CITED

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2. Demmig-Adams B (1990) Carotenoids and photoprotection inplants: a role for the xanthophyll zeaxanthin. Biochim BiophysActa 1020: 1-24

3. Guillard RRL, Ryther JH (1962) Studies on marine planktondiatoms. I. Cyclotella nana (Hustedt) and Detonula confervaceae(Cleve). Can J Microbiol 8: 229-239

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5. Holmes JJ, Weger HG, Turpin DH (1989) Chlorophyll a fluo-rescence predicts total photosynthetic electron flow to CO2 orN03-/NO2- under transient conditions. Plant Physiol 91:331-337

6. Horton P, Hague A (1988) Studies on the induction of chloro-phyll fluorescence in isolated barley protoplasts. IV. Resolutionof non-photochemical quenching. Biochim Biophys Acta 932:107-115

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